Structural comparison of differently glycosylated forms of acid--glucosidase, the defective enzyme in Gaucher disease

Abstract

Gaucher disease is caused by mutations in the gene encoding acid-beta-glucosidase. A recombinant form of this enzyme, Cerezyme, is used to treat Gaucher disease patients by ;enzyme-replacement therapy'. Crystals of Cerezyme after its partial deglycosylation were obtained earlier and the structure was solved to 2.0 A resolution [Dvir et al. (2003), EMBO Rep. 4, 704-709]. The crystal structure of unmodified Cerezyme is now reported, in which a substantial number of sugar residues bound to three asparagines via N-glycosylation could be visualized. The structure of intact fully glycosylated Cerezyme is virtually identical to that of the partially deglycosylated enzyme. However, the three loops at the entrance to the active site, which were previously observed in alternative conformations, display additional variability in their structures. Comparison of the structure of acid-beta-glucosidase with that of xylanase, a bacterial enzyme from a closely related protein family, demonstrates a close correspondence between the active-site residues of the two enzymes.

Full text

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Acta Crystallographica Section D
Biological
Crystallography
ISSN 0907-4449
Editors: E. N. Baker and Z. Dauter
Structural comparison of differently glycosylated forms of
acid-
¬
-glucosidase, the defective enzyme in Gaucher disease
Boris Brumshtein, Mark R. Wormald, Israel Silman, Anthony H. Futerman and Joel
L. Sussman
Copyright © International Union of Crystallography
Author(s) of this paper may load this reprint on their own web site provided that this cover page is retained. Republication of this article or its
storage in electronic databases or the like is not permitted without prior permission in writing from the IUCr.
Acta Cryst.
(2006). D62, 1458–1465 Brumshtein
et al.
¯
Acid-
¬
-glucosidase

research papers
1458 doi:10.1107/S0907444906038303 Acta Cryst. (2006). D62, 1458–1465
Acta Crystallographica Section D
Biological
Crystallography
ISSN 0907-4449
Structural comparison of differently glycosylated
forms of acid-b-glucosidase, the defective enzyme
in Gaucher disease
Boris Brumshtein,
a
Mark R.
Wormald,
b
Israel Silman,
c
Anthony H. Futerman
d
and
Joel L. Sussman
a
*
a
Department of Structural Biology, Weizmann
Institute of Science, Israel,
b
Oxford
Glycobiology Institute, Department of
Biochemistry, University of Oxford,
Oxford OX1 3QU, England,
c
Department of
Neurobiology, Weizmann Institute of Science,
Israel, and
d
Department of Biological Chemistry,
Weizmann Institute of Science, Israel
Correspondence e-mail:
joel.sussman@weizmann.ac.il
#2006 International Union of Crystallography
Printed in Denmark – all rights reserved
Gaucher disease is caused by mutations in the gene encoding
acid--glucosidase. A recombinant form of this enzyme,
Cerezyme
1
, is used to treat Gaucher disease patients by
‘enzyme-replacement therapy’. Crystals of Cerezyme
1
after
its partial deglycosylation were obtained earlier and the
structure was solved to 2.0 A
˚resolution [Dvir et al. (2003),
EMBO Rep. 4, 704–709]. The crystal structure of unmodified
Cerezyme
1
is now reported, in which a substantial number of
sugar residues bound to three asparagines via N-glycosylation
could be visualized. The structure of intact fully glycosylated
Cerezyme
1
is virtually identical to that of the partially
deglycosylated enzyme. However, the three loops at the
entrance to the active site, which were previously observed in
alternative conformations, display additional variability in
their structures. Comparison of the structure of acid--gluco-
sidase with that of xylanase, a bacterial enzyme from a closely
related protein family, demonstrates a close correspondence
between the active-site residues of the two enzymes.
Received 4 July 2006
Accepted 19 September 2006
PDB Reference: acid-
-glucosidase, 2j25, r2j25sf.
1. Introduction
Gaucher disease, the most common lysosomal storage
disorder (Futerman & van Meer, 2004), is caused by mutations
in the gene encoding acid--glucosidase (glucocerebrosidase,
GlcCerase; EC 3.2.1.45; Beutler & Grabowski, 2001), resulting
in intracellular accumulation of glucosylceramide (GlcCer).
Fig. 1 shows the reaction catalyzed by GlcCerase. GlcCerase is
a 497 amino-acid residue enzyme with a molecular weight of
62 kDa (Horowitz et al., 1989; Grabowski et al., 1990).
Mutations in GlcCerase diminish activity either by reducing
catalytic activity or by reducing its lysosomal concentration. In
the former case the mutations affect turnover number,
substrate affinity and/or activator binding and in the latter
they compromise folding in the endoplasmic reticulum,
resulting in proteasomal degradation of the protein (Sawkar et
al., 2005). GlcCerase is activated in lysosomes by saposin C
(SapC; Bruhn, 2005; Vaccaro et al., 1997), although neither the
mechanism of activation nor the precise role of SapC are well
understood (Bruhn, 2005).
We previously determined the three-dimensional structure
of Cerezyme
1
(Premkumar et al., 2005; Dvir et al., 2003), a
recombinant form of GlcCerase that is used in enzyme-
replacement therapy (ERT) for Gaucher disease patients
(Jmoudiak & Futerman, 2005). The protein consists of three
non-contiguous domains, with the catalytic site located in
domain III (residues 76–381 and 416–430), a (/)
8
(TIM)
barrel. Although the function of the two non-catalytic
domains is unknown, mutations that cause Gaucher disease
are found in all three domains.
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There are five putative glycosylation sites in GlcCerase, four
of which are believed to be occupied (Asn19, Asn59, Asn146
and Asn270; Grace et al., 1994). In order to target GlcCerase
to macrophages, the main cell type affected in Gaucher
disease (Jmoudiak & Futerman, 2005), and to enhance inter-
nalization by mannose receptors on the surfaces of the
macrophages (Brady, 2006), production of Cerezyme
1
involves the sequential deglycosylation of GlcCerase,
using -neuraminidase, -galactosidase and -N-acetyl-
glucosaminidase, to expose terminal mannose residues,
leaving the core glycan, an oligosaccharide which consists of
five sugars, namely two N-acetylglucosamines and three
mannoses.
In our previous structure determinations (Premkumar et al.,
2005; Dvir et al., 2003), Cerezyme
1
was partially deglycosy-
lated prior to crystallization using N-glycosidase F, which
removes carbohydrate chains by cleaving the amide bonds
between Asn residues and N-acetylglucosamine (GlcNAc;
Han & Martinage, 1992), but does not necessarily remove all
carbohydrate chains from native proteins; a similar protocol
was used to obtain another recently reported Cerezyme
1
structure (PDB code 2f61; Liou et al., 2006).
In order to alleviate concerns that partial deglycosylation
might alter its three-dimensional structure, we have now
solved the structure of intact Cerezyme
1
(GCase) obtained
without N-glycosidase F treatment. The crystals display the
same space group as the partially deglycosylated Cerezyme
1
(pDG-GCase); moreover, there are no fundamental differ-
ences between the two structures, although some novel
conformations are observed in the lid region around the active
site (Premkumar et al., 2005). In addition, we demonstrate that
GlcCerase bears a strong structural similarity in its active-site
region to xylanase (Larson et al., 2003), a glycoside hydrolase
that shows the highest sequence similarity to GlcCerase
among structures deposited in the PDB.
2. Experimental procedures
2.1. Crystallization
Cerezyme
1
(GCase; Genzyme Corporation), obtained
from patient leftovers, was dissolved in 100 mMNaCl, 50 mM
2-(4-morpholino)ethanesulfonic acid (MES) pH 5.5 at a
concentration of 1–2 mg ml
1
. The sample was washed with
the same buffer and concentrated to 4–5 mg ml
1
in a
Centricon device using a filter with a cutoff size of 30 kDa.
GCase crystals were obtained by microbatch crystallization
using a Douglas Instruments IMPAX I-5 robot. The crystal-
lization solution contained a 1:1 ratio of the concentrated
enzyme solution and 2 M(NH
4
)
2
SO
4
,0.1MBis-Tris pH 5.5.
Crystallization was performed under oil (D’Arcy et al., 2003)
for 5–14 d at 293 K. Data were collected on beamline ID23eh1
at the ESRF synchrotron facility in Grenoble, France. Crystals
were mounted and flash-cooled at 100 K. X-ray diffraction
images were processed using XDS and XSCALE (Kabsch,
1993). Reflections were converted to a format suitable for
REFMAC5 (Murshudov et al., 1997) using XDSCONV and
processed in CCP4 (Collaborative Computational Project,
Number 4, 1994). Table 1 summarizes data collection and
processing.
2.2. Structure determination and refinement
Initial phases were obtained by molecular replacement
using Phaser (McCoy et al., 2005) software. Molecule Aof
pDG-GCase (PDB code 1ogs; Dvir et al., 2003) was used as a
starting model for molecular replacement. REFMAC5
(Murshudov et al., 1997) was used for refinement and Coot
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Acta Cryst. (2006). D62, 1458–1465 Brumshtein et al. Acid--glucosidase 1459
Figure 1
Reactions catalyzed by GlcCerase and xylanase.
Table 1
Data-collection statistics for GCase.
Values in parentheses are for the highest resolution shell (2.97–2.9 A
˚).
Radiation wavelength (A
˚) 0.9759
Temperature (K) 100
Space group C222
1
Unit-cell parameters (A
˚)a= 108.6, b= 280.8, c= 91.0
Resolution range (A
˚) 40–2.9
No. of observed reflections 228034 (16759)
No. of unique reflections 30978 (2227)
Completeness (%) 98.7 (98.5)
hIi/(I) 13.75 (5.3)
R
mrgd-F
(%) 10.6 (27.0)
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(Emsley & Cowtan, 2004) was used to build the model and fit
it into the electron density. During refinement and model
building, twofold noncrystallographic symmetry was applied,
except for regions where different conformations were clearly
detectable (Kleywegt, 1996). Residues 312–319 (loop 3) in
molecule Bof the GCase model did not fit the electron
density. Consequently, they were omitted from the refinement
and rebuilt into a new conformation.
Electron-density maps revealed sugar residues attached to
some of the Asn residues. Sugars were modelled into electron
density according to the putative oligosaccharide sequences
reported by the Genzyme Corporation for Cerezyme
1
(US
patent Nos. 5236838 and 5549892; Murray, 1987). The con-
formations and angles of the oligosaccharides were confirmed
with PDB-CARE (Lutteke & von der Lieth, 2004) and CARP
(Lutteke et al., 2005). Additional sugars, SO2
4ions and water
molecules were added with improvement of electron-density
maps and phases in subsequent refinement. Refinement
results are summarized in Table 2.
2.3. Comparison of the structures of GCase and xylanase
ABLAST search for sequences in the PDB (Berman et al.,
2000) was performed using the GCase sequence (Horowitz et
al., 1989). The best fit was achieved for xylanase (EC 3.2.1.8;
PDB code 1nof), a member of glycoside hydrolase family 5
(Larson et al., 2003). According to BLASTP (Altschul et al.,
1997), the expectation value is 5 10
3
, with 18% identity and
41% similarity. The crystal structure of xylanase reveals a fold
similar to that of GCase. Therefore, 1nof, which was deter-
mined to 1.42 A
˚resolution (Larson et al., 2003), was used to
cross-validate structural features of GCase (Keen et al., 1996).
2.4. Validation and deposition
The final models and structure factors of GCase were
validated with PROCHECK (Laskowski et al., 1993) and
deposited in the PDB with code 2j25.
2.5. Modelling glycans on pDG-GCase
Glycan modelling was performed on a Silicon Graphics Fuel
workstation using INSIGHTII and DISCOVER software
(Accelrys Inc., San Diego, USA). N-linked glycan structures
were generated using the database of glycosidic linkage
conformations (Wormald et al., 2002) and in vacuo energy
minimization to relieve unfavourable steric interactions. The
Asn–GlcNAc linkage conformations and analysis of the Asn
side-chain conformations were based on the observed range of
crystallographic values (Petrescu et al., 2004). The nomen-
clature for the Asn side-chain torsion angles is 
1
=N—C

—
C

—C

,
2
=C

—C

—C

—N

. Figures were produced with
PyMol (http://www.pymol.org).
3. Results
3.1. Crystallization
Previous attempts to obtain Cerezyme
1
crystals without
partial deglycosylation were unsuccessful (Dvir et al., 2003;
Liou et al., 2006; Premkumar et al., 2005). In the current study,
using a much larger screen of crystallization conditions (956
different conditions, excluding optimizations), employing the
microbatch technique under oil (Chayen, 1998) and utilizing a
Douglas Instruments IMPAX I-5 crystallization robot at room
temperature, we were finally able to obtain crystals of Cere-
zyme
1
without prior deglycosylation (GCase). To improve
diffraction, the conditions which produced the best crystals
were chosen for further optimization. GCase crystallizes in the
same space group as previously reported for pDG-GCase, i.e.
C222
1
, and with similar unit-cell parameters.
Analysis of the two structures indicates that all glycosyl-
ation sites are adjacent to empty cavities in the crystal, thus
allowing placement of the sugars in these spaces without
generating steric clashes that would hinder crystallization. The
asymmetric unit of pDG-GCase was shown to contain two
copies of the GlcCerase molecule, molecules Aand B, which,
although very similar, are not completely identical to each
other in conformation (Dvir et al., 2003; Premkumar et al.,
2005). They also differ in the number of sugar molecules for
which electron density can be assigned (Dvir et al., 2003).
Some of the glycoside chains from adjacent asymmetric units
make contacts with each other. These contacts stabilize and
order those oligosaccharide chains in the crystal, thus making
them visible in the electron-density maps.
The structure of GCase was refined to 2.9 A
˚resolution.
Some conformational differences were observed between
molecules Aand Bin GCase and the corresponding molecules
in pDG-GCase (PDB codes 1ogs and 1y7v; Dvir et al., 2003;
Premkumar et al., 2005). The changes seen are mainly in loops
near the active site.
3.2. Glycosylation of GCase
Owing to crystal-packing constraints, the glycans in mole-
cule Bare less mobile than those in molecule A(Fig. 2a). Thus,
in molecule Ba core glycan chain containing five sugar resi-
dues is seen attached to Asn19, three sugars are seen attached
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1460 Brumshtein et al. Acid--glucosidase Acta Cryst. (2006). D62, 1458–1465
Table 2
Refinement statistics for the GCase structure.
Resolution range of refinement (A
˚) 29.6–2.9
R
work
† (%) 21.5
R
free
‡ (%) 27.3
Monomers per ASU 2
No. of different NCS groups 2
Average Bfactor (A
˚
2
) 33.6
R.m.s. deviations from ideal values
Bond lengths (A
˚) 0.015
Bond angles () 1.590
Torsion angles () 6.627
Estimated coordinate error
E.s.d.§ from Luzzati plot 0.38
ESU}based on Rfree (A
˚)0.42
ESU based on maximum likelihood (A
˚) 0.31
ESU for Bvalues based on maximum likelihood (A
˚
2
) 17.01
Ramachandran outliers (%) 0.2
†R
work
=PjFojjFcj=PjFoj, where F
o
denotes the observed structure-factor
amplitude and F
c
the structure-factor amplitude calculated from the model. ‡ R
free
is
for 5% of randomly chosen reflections excluded from the refinement. § E.s.d.,
estimated standard deviation. }ESU, estimated standard uncertainty
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to Asn59, two to Asn146 and none to Asn270 (Table 3). In
molecule Aonly two sugars can be detected on Asn19, one on
Asn146 and none on Asn59 and Asn270. The glycans attached
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Acta Cryst. (2006). D62, 1458–1465 Brumshtein et al. Acid--glucosidase 1461
to residue Asn19-B(i.e. Asn19 of molecule B) and to Asn59-B
of a symmetry-related molecule are in contact with each other
(Fig. 2b), causing the glycan chains to become ordered and, as
a consequence, visible in the electron-density map. The two
sugar moieties on the glycan of Asn146-Bdo not make crystal
contacts, consistent with the low number of sugar residues
visible on Asn146 (Fig. 2c). The fact that we do not detect any
sugars on residue Asn59-Amay be a consequence of the
flexibility of this glycan in the crystal. Residues 59–64 adopt
two different conformations in molecules Aand Bowing to
different crystal contacts.
3.3. Structural characteristics of GCase
Some conformational differences, as distinguished by
differences in ’and angles (Kleywegt, 1996), were detected
between molecules Aand Bof the asymmetric unit of GCase
(Fig. 3).
3.3.1. Lid at the entrance to the active site. We previously
described two loops in pDG-GCase whose conformations
control access to the active site (Dvir et al., 2003; Premkumar
et al., 2005). In molecule Aof GCase, we now detect confor-
mational differences in an additional loop, loop 3 (residues
312–319; Table 4), that arise in conjunction with changes in the
-sheet structure of residues 341–344, which are located
adjacent to the catalytic residue Glu340 (Fig. 4). The re-
arrangement of loop 3 consists of side-chain flips of both
Trp312 and Trp378 (Fig. 5), which are associated with the
transition of the conformations of residues 312–319 and 341–
344 from a loop and a -strand, respectively, to coils. However,
these differences do not affect the conformation of the cata-
lytic residues, since the distances between the side chains of
Glu235 and Glu340 remain similar (Table 5). Moreover, the
Figure 2
Packing of glycans in the GCase crystal. (a) Glycans in the cavities between the symmetry-related molecules in the crystal. Molecule Bof the asymmetric
unit is shown in blue and the symmetry-related molecules are shown in yellow. The regions labelled Band Care magnified in (b) and (c), respectively. (b)
Magnification of a view of the sugars bound to Asn59 (blue) and to Asn19 on a symmetry-related molecule (yellow). (c) Magnification of a view of the
sugars bound to Asn146 (blue).
Table 3
Number of bound sugar residues observed in the crystal structures of
GlcCerase.
Asn19 Asn59 Asn146 Asn270
2j25-A201 0
2j25-B532 0
1ogs-A100 0
1ogs-B200 0
1y7v-A200 0
1y7v-B200 0
2f61-A100 0
2f61-B200 0
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conformation of His311 (see below) does not change. In
contrast, we detected no new conformations of loops 1–3 in
molecule Brelative to 1ogs and 1y7v, but did detect a novel
combination of previously observed conformations of the
loops.
3.3.2. Active site. Table 5 shows that the distances between
Glu340 and Glu235, the catalytic site residues of GCase, are
5A
˚, which is consistent with a retaining mechanism (Davies
& Henrissat, 1995) of catalytic activity. It is also seen from this
table that the average distances between O
"1
O
"1
and
O
"2
O
"2
are similar in all the crystal structures reported to
date. The differences in distances between individual
carboxylate groups of the catalytic residues of 2j25 and of 1ogs
and 1y7v arise from changes in 
2
torsion angles. Since the
acidic pK
a
of the pH-activity profile is around pH 4.7 (Liou et
al., 2006) and the pH values for the crystal structures exam-
ined here lie on either side of this pK
a
value (Dvir et al., 2003;
Premkumar et al., 2005), it is plausible that the observed
differences are related to the protonation states of the active-
site carboxyl groups. Thus, 1ogs and 1y7v, which were both
crystallized at pH 4.6, would be expected to have both
carboxyl groups protonated. This would explain both the
greater inter-residue distances and changes in 
2
torsion
angles relative to 2j25. The latter, having been crystallized at
pH 5.5, would be expected to have only one carboxyl group
protonated, which should allow closer approach of the two
carboxylates.
3.3.3. Anion-binding sites. In GCase, there are two clusters
of SO2
4ions, which were previously detected in 1ogs and 1y7v
but not analyzed in detail (Fig. 6a); only one of these clusters
was described in 2f61 (Liou et al., 2006). Each cluster contains
2–3 SO2
4ions. One cluster is located close to residues 12, 44,
45, 353 and 356–358, near the active site (Fig. 6a). The second
is near residues 79, 228, 277 and 306. Since the negative charge
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1462 Brumshtein et al. Acid--glucosidase Acta Cryst. (2006). D62, 1458–1465
Figure 3
A’, difference plot between main-chain angles in molecules of the
asymmetric unit of GCase (PDB code 2j25). The differences between
angles are constrained to be between 180and +180.
Table 4
Conformational classification of loops L1, L2 and L3 in the various
GlcCerase crystal structures.
Loop L3, 312–319 L1, 341–347 L2, 393–399
Conformation
Open† 2j25-B2j25-A, 2j25-B2j25-B
1ogs-A, 1ogs-B1ogs-A1ogs-B
1y7v-A, 1y7v-B1y7v-A, 1y7v-B
2f61-A, 2f61-B2f61-A2f61-B
Closed† 2j25-A2j25-A
1ogs-B1ogs-A
1y7v-A, 1y7v-B
2f61-B2f61-A
† The assignment of loop conformations as open or closed is based on the criteria
established for 1y7v (Premkumar et al., 2005) and referred to in the discussion.
Figure 4
Conformational classification of loops 1, 2 and 3 (Kleywegt, 1996). The
structures of the loops of 2j25-A(GCase) are in yellow, 2j25-Bin green,
1y7v (pDG-GCase) in magenta, 1ogs-A(pDG-GCase) in orange, 1ogs-B
in red, 2f61-A(pDG-GCase) in black and 2f61-Bin grey. Catalytic
residues are in red.
Table 5
Interatomic distances between side chains of catalytic residues.
Distances are shown in A
˚for the following residues: Glu235–Glu340
(O
"1
O
"1
), Glu235–Glu340 (O
"2
O
"2
) and Glu235–His311 (O
"2
N
1
).
The average distance is between the carboxyl O atoms of the catalytic
glutamates. In the case of 1nof, the distances are measured between residue
Glu165 and residues Glu253 and His230.
O
"1
O
"1
O
"2
O
"2
Average
distance
Closest
approach O
"2
N
1
1ogs-A5.9 4.2 5.05 4.2 2.7
1ogs-B6.2 4.3 5.25 4.3 2.8
1y7v-A6.2 4.0 5.10 4.0 2.8
1y7v-B6.1 3.9 5.00 3.9 2.9
2j25-A4.6 4.2 4.40 4.2 3.2
2j25-B4.9 4.1 4.50 4.1 3.1
2f61-A5.0 5.2 5.10 4.7 3.4
2f61-B6.3 3.9 5.10 3.9 2.6
1nof 5.8† 4.2† 5.00 4.2 2.6
† The numbering of O
"1
and O
"2
is arbitrary and the assignment in 1nof was therefore
altered so as to correspond to the numbering in the other structures.
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of SO2
4is similar to that of the negatively charged phospho-
lipids required for optimal GCase activity in vivo (Grace et al.,
1994), we suggest that the SO2
4cluster adjacent to the
active site may be involved in the membrane association of
GlcCerase (Fig. 6b).
3.4. Comparison of the active sites of GCase and xylanase
We compared the structure of GCase with that of xylanase
(PDB code 1nof; Larson et al., 2003), a member of glycoside
hydrolase family 5, which shows the highest sequence identity
to GlcCerase of all structures in the PDB. In order to cross-
validate the positioning and distances of the catalytic residues,
the GCase structure was aligned with that of xylanase (Fig. 7).
Comparison of the active sites was attained by aligning the
catalytic residues: Glu235 and Glu340 in GCase and Glu165
and Glu253 in xylanase. This alignment gave nine identical
and two similar residues within 5 A
˚of the catalytic residues,
which align virtually identically. Only one residue, Tyr313 in
GCase, which corresponds to Tyr232 in xylanase, shows
conformational variability.
Based on the structural and sequence alignment, His311 in
GCase corresponds to His230 in xylanase. The distances
between His311 and Glu235 in GCase and between His230
and Glu165 in xylanase are compatible with hydrogen
bonding. This conserved histidine residue may introduce
additional hydrogen-bond coordination between the catalytic
residues and thus play a role in stabilizing the active sites of
both GCase and xylanase.
4. Discussion
The major conclusion of the current study is that partial
deglycosylation of Cerezyme
1
(pDG-GCase) by N-glycosi-
dase F has no major effect on the conformation of GlcCerase,
thus validating our previous structural analyses of crystals
obtained after N-glycosidase F treatment.
We observe a significant number of Asn-linked glycans in
the GCase structure. The packing of the protein molecules in
the crystal allows the insertion of sugar chains into the free
cavities, which are sufficiently large to accommodate the
glycans without introducing steric clashes with symmetry-
related molecules. We are able to observe two large segments
of two glycan chains, those attached to Asn19-Band Asn59-B,
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Acta Cryst. (2006). D62, 1458–1465 Brumshtein et al. Acid--glucosidase 1463
Figure 6
Putative membrane binding sites in crystal structures of GlcCerase. Catalytic residues are shown in red and loops 1–3 in green. Substrate binding was
modelled according to Dvir et al. (2003) and shown in yellow. (a) Some of the sulfates are shown in space-filling representation in red. (b) Side-on view
[rotated 90around the horizontal axis relative to (a)] of a putative mode of binding of GlcCerase to negatively charged phospholipids.
Figure 5
Conformational changes in Trp312 and Trp378 and movements in the
backbone near the active site. Yellow, 2j25-A; green, 2j25-B; orange, 1ogs-
A; red, 1ogs-B; black, 2f61-A; grey, 2f61-B; magenta, 1y7v; catalytic
residue in red.
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which are in contact with each other and thus mutually
stabilize each other’s conformations. It was previously
reported that the visualization of a sequence of seven sugars of
a glycan chain in Erythrina corallodendron lectin could simi-
larly be ascribed to its immobilization by a symmetry-related
molecule, whereas only chitobioses could be detected on
chains that were not stabilized by such intermolecular inter-
actions (Shaanan et al., 1991).
The observation of sugar residues attached to Asn19 in
pDG-GCase suggested that this residue may be inaccessible to
enzymatic cleavage by N-glycosidase F. This was verified by
molecular modelling, which was performed to determine
whether all four N-linked sites are available for glycosylation
in the monomer and whether the glycans modelled at these
sites block access to the active site. Modelling of the crystal
packing for pDG-GCase showed that except for Asn19, the
putative glycosylation sites could not be occupied with the
observed Asn side-chain conformations. According to the
modelling, a glycan attached to Asn146-Ain pDG-GCase with
the observed side-chain torsion angles (Petrescu et al., 2004)
would clash sterically with a glycan attached to Asn146-B,
indicating that both sites cannot be occupied simultaneously.
Similarly, a core glycan chain modelled on Asn19-Bwould
clash with a modelled glycan on Asn59 of the adjacent B
chain. Thus, the fact that well resolved electron density was
seen for a glycan on Asn19-Bin pDG-GCase implies that
Asn59 must be unoccupied. The presence of glycans on Asn59
and Asn146 in the new GCase structure causes changes in the

1
and 
2
torsion angles of the Asn side chains (Petrescu et al.,
2004), thus eliminating steric clashes.
Despite the differences in crystallization conditions and
glycosylation patterns, there are only minor differences
between the pDG-GCase and GCase structures, which have a
similar asymmetric unit and crystal packing, as confirmed by
the low r.m.s. deviations (Table 6). However, several small but
important differences were detected in the conformation of
the lid in molecule Aof GCase, encompassed by loops 1–3
(Premkumar et al., 2005). Importantly, these differences
cannot be ascribed to glycosylation, since molecule Bin the
GCase structure shows conformations of loops 1–3 that are
identical to those seen in pDG-GCase. Molecule Ain GCase
displays some additional changes near the active site relative
to pDG-GCase, in residues Trp312, Trp378 and 341–344. Most
significant are the changes in Trp312 and Trp378, whose side-
chain conformations depend on the conformation of loop 3; as
a result, the conformations of residues 341–344 are also
altered. It should be stressed that even though these loops
control access to the active site, substrate binding must
produce a conformational change in the loops in order to
allow the process to occur without the introduction of steric
clashes, similar to that seen in glycosyl transferases (Qasba et
al., 2005).
Comparison of the catalytic residues Glu235 and Glu340 in
the various GlcCerase structures reveals minor positional
differences, some of which arise from changes in the torsion
angles of Glu235. The side-chain conformation of Glu235
depends on interactions with surrounding residues via
hydrogen bonds. This may explain the differences in the
distances between Glu235 and Glu340 in the various struc-
tures solved to date (Table 5). Moreover, it was recently
implied that the structure of pDG-GCase may not be valid
owing to the loss of activity caused by N-glycosidase F treat-
ment (Liou et al., 2006). That this is not the case is supported
by the similarity in distances between Glu235 and Glu340
(1ogs, 1y7v and 2j25 versus 2f61). Moreover, we have shown
that pDG-GCase is fully catalytically active (Premkumar et al.,
2005). Unfortunately, no data are presented to document the
purported loss of activity produced by N-glycosidase F treat-
ment (Liou et al., 2006). In addition, considerable concern
remains about the lack of correlation between the electron
density and the reported coordinates in some regions of 2f61
(Liou et al., 2006). Irrespective of these concerns, the struc-
tures of 2f61 and of 1ogs, 1y7v and 2j25 are all very similar,
with the minor exceptions noted above with respect to 2j25.
In summary, both the pDG-GCase and GCase structures
reported by us are fully compatible with the catalytic
research papers
1464 Brumshtein et al. Acid--glucosidase Acta Cryst. (2006). D62, 1458–1465
Table 6
Comparison of GlcCerase monomers in various crystal structures.
R.m.s. deviations are shown in A
˚.
1ogs-A1ogs-B1y7v-A1y7v-B2f61-A2f61-B
2j25-A0.35 0.37 0.34 0.34 0.37 0.39
2j25-B0.37 0.34 0.33 0.31 0.39 0.35
2f61-A0.19 0.33 0.30 0.33
2f61-B0.31 0.17 0.30 0.28
1y7v-A0.25 0.26
1y7v-B0.27 0.24
Figure 7
Comparison of the active sites of GCase and xylanase. Residues within a
5A
˚radius of the active site are displayed. Yellow, 2j25-A(GCase); green,
2j25-B; blue, 1nof (xylanase); orange, 1ogs-A(pDG-GCase); red, 1ogs-B;
black, 2f61-A(pDG-GCase); grey, 2f61-B. The first number refers to
residue in GCase and the second number to the corresponding residue in
xylanase.
electronic reprint

mechanism of GlcCerase and provide the molecular tools for
detailed structure–function analysis, which may lead to
improved GlcCerase for use in enzyme-replacement therapy.
This work was supported by the Horowitz Foundation, the
National Gaucher Foundation, the Oxford Glycobiology
Institute, the Yeda fund of the Weizmann Institute, the
Divadol Foundation, the Bruce Rosen Foundation, the
Kalman and Ida Wolens Foundation, the Jean and Jula
Goldwurm Memorial Foundation, the Kimmelman Center for
Biomolecular Structure and Assembly, the Benoziyo Center
for Neuroscience and the Minerva Foundation. We thank the
staff at beamline ID23eh1 at the ESRF synchrotron in
Grenoble for assistance during data collection, Dr Orly Dym
for help in examining the structures and Dr Harry Greenblatt
and Yehudit Hasin for commenting on the manuscript. JLS is
the Morton and Gladys Pickman Professor of Structural
Biology and AHF is the Joseph Meyerhoff Professor of
Biochemistry.
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